ABSTRACT Due to the lack of widespread 2nd-tier screening tests, newborn screening providers and parents struggle to understand the ramification of genetic conditions detected during the neonatal period. This is especially true in newborn screening for Lysosomal Storage Diseases (LSDs) such as Pompe and Mucopolysaccharidosis Type I (MPS I), where pseudodeficient variants result in a lack of enzyme activity in the newborn dried blood spot (DBS) screening test, but present with normal endogenous enzyme activity and with no overt disease manifestations. Current screening algorithms do not routinely identify or provide molecular genetic etiology for LSDs because the genotype is not routinely monitored. However, frequency of pseudodeficiency mutations can be as high as 4% in certain populations and can be a significant source of false positive (+) results. Enhanced sensitivity and specificity in newborn screening for Pompe and MPS I is essential, but extremely challenging. Correct diagnosis of a disease, and its underlying biochemical and molecular basis, is not only critical for successful differential diagnosis and treatment early in life but it is also important in reducing anxiety in parents and unnecessary burdens on follow-up programs. Current gene sequencing methods including genome scale sequencing [Whole Exome and Whole Genome (WES and WGS, respectively)] used for confirmatory diagnosis are impractical in newborns and do not scale. A targeted next-generation sequencing (tNGS) panel method to address deficiencies in current testing can be used as a routine 2nd-tier newborn screen to significantly improve sensitivity and specificity. The panel designs of single genes are impractical for scaling in screening conditions due to the rare nature of LSDs and maintenance of multiple assays. Genome scale sequencing approaches are not comprehensive across non coding regions (introns, promoters, etc.) with gaps in coverage or high costs that are prohibitive for screening paradigms. We will create a comprehensive newborn specific gene panel for LSDs by establishing sample collection and processing workflows more appropriate for newborns. We will demonstrate the power of tNGS for reducing false (+) results by identifying clinical variants and pseudodeficiency mutations in Pompe and MPS I. Our goal is to develop an assay with high sensitivity and specificity. We will demonstrate the value of the assay when used as a 2nd-tier screen following 1st-tier enzyme analysis via the Baebies' FDA cleared SEEKER device for high throughput newborn screening of LSDs. We will use the results to develop an algorithm that can be used by newborn screening programs for accurate interpretation of screening results. These studies will eventually allow CLIA/CAP validation of our tNGS methodology and permit us to offer our screening panel for LSDs on a commercial basis. Our approach has the potential to rapidly and simultaneously screen for hundreds of other LSD or newborn conditions with future development. Early identification of many of these additional disorders is critical for rapid and appropriate management.